Introduction

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There is considerable evidence that tolerance can develop during an initial dose of EtOH and that this acute tolerance influences the apparent acute behavioral sensitivity of laboratory animal models to EtOH (Goldstein, 1983; Laverty, 1989; Littleton, 1980; Mellanby, 1919). Keir and Deitrich (1990) compared blood EtOH levels in LS and SS mice at the regaining of righting reflex over a range of systemic EtOH doses and concluded that tolerance to EtOH-induced ataxia can occur within minutes of the EtOH injection. In contrast, no evidence of acute tolerance was found in earlier studies either using a dose-response paradigm that was similar to that used by Keir and Deitrich, except that higher EtOH doses were used (Smolen and Smolen, 1987), or comparing blood EtOH concentrations at loss and recovery of the righting response (Tabakoff and Ritzmann, 1979; Tabakoff et al., 1980). However, the LS and SS mice in these latter studies lost righting response >5 min after EtOH administration, and Gill et al. (1993) found evidence that acute tolerance to EtOH-induced ataxia on Rotorod performance developed within 3 to 5 min in SS mice. Thus, the mechanism of EtOH action in the central nervous system may have desensitized before the earliest measurements made in the aforementioned studies of righting response. Indeed, Allan and Harris (1987) reported that tolerance developed to EtOH augmentation of GABA_A-mediated chloride flux in cerebellar microsacs within the first 5 min of a systemic EtOH dose. We previously found that this GABA mechanism mediates EtOH-induced depressions of cerebellar Purkinje neuron firing rate (Freund et al., 1993; Lin et al., 1993; Palmer and Hoffer, 1990), and we have reported that RANT, a cellular desensitization to this electrophysiological effect of locally applied EtOH, develops within the first few minutes after the initial EtOH application in SS mice (Sorensen et al., 1980), as well as in LAS rats (Palmer et al., 1992). However, the time course of the development of and recovery from RANT has not been previously characterized.

Differences in Purkinje neuron sensitivity to the acute depressant effects of EtOH on spontaneous discharge between the LS and SS mouse populations correlate with behavioral sensitivity to EtOH-induced ataxia in those animals (Sorensen et al., 1980). Like LS and SS mice, HAS and LAS rats have been selectively bred for susceptibility to EtOH-induced ataxia (Palmer et al., 1987; Spuhler et al., 1990), and we have found that this behavioral sensitivity correlates with the sensitivity of cerebellar Purkinje neurons to the depressant effects of EtOH in both replicates of each selected rat line (Palmer et al., 1992). Furthermore, these two phenotypes show a striking genetic correlation among inbred strains of rats and mice (Johnson et al., 1985; Palmer et al., 1987; Spuhler et al., 1982). Behavioral data from several laboratories suggest that the development of acute EtOH tolerance contributes to the acute EtOH sensitivity differences observed between lines of animals, such as these, that are bred for their behavioral responsiveness to EtOH. Thus, the EtOH-insensitive SS mice and AT (alcohol-tolerant) selected lines of rats develop acute tolerance to the ataxia-producing effects of repeated acute EtOH doses more rapidly than do their EtOH-sensitive counterparts, LS mice and ANT (alcohol-nontolerant) rats (Keir and Deitrich, 1990; Le and Kiianmaa, 1989; Parsons et al., 1982). We previously reported that EtOH-insensitive SS mice, but not EtOH-sensitive LS mice, express RANT to the depressant effects of EtOH on Purkinje neuron firing rate (Sorensen et al., 1980). More recently, we found that LAS and HAS rats also differentially express RANT to the depressant effects of EtOH on Purkinje neurons (Palmer et al., 1992). Although RANT was observed from more than half of EtOH-naive Purkinje neurons in EtOH-insensitive LAS rats, this phenomenon was observed from only 5% of these cells in EtOH-sensitive HAS rats. Previously published work on LS and SS mice clearly suggests that the EtOH insensitivity of SS mice is mediated not only by low initial sensitivity to EtOH but also by the development of tolerance to EtOH effects within the first few minutes of the initial EtOH exposure (Keir and Deitrich, 1990). Similarly, we found that the neuronal insensitivity of LAS rats to EtOH is due not only to a lower sensitivity of these cells to the initial depressant effect of EtOH but also to the rapid development of acute tolerance to this neuronal EtOH effect (Palmer et al., 1992).

In the present investigation, we studied RANT to the depressant effects of EtOH in HAS and LAS rats. These animals were selectively bred for 21 generations from genetically heterogeneous N/NIH rats (Hansen and Spuhler, 1984; Spuhler et al., 1990), and selection was based on acute sensitivity or insensitivity, respectively, to EtOH-induced loss of righting response. The present study characterizes the electrophysiological development of and recovery from RANT to the depressant effects of locally applied EtOH in Purkinje neurons of LAS and HAS rats and explores the role of this phenomenon in the neuronal sensitivity differences to acute EtOH between these two selected rat lines. Furthermore, we previously found that there is a greater expression of RANT in LAS rats and SS mice than in HAS rats or LS mice (Palmer et al., 1992; Sorensen et al., 1980). However, the HAS rats and LS mice were not exposed to the higher doses of EtOH that are required to depress the firing of Purkinje neurons in the less sensitive LAS rats or SS mice. Thus, we standardized the dose of EtOH to that which caused maximal neuronal tolerance to EtOH in LAS neurons from a single local application, and we then used this dose to characterize RANT in both LAS and HAS Purkinje neurons.

	Methods

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Forty-one HAS and 62 LAS rats from the 21st generation of selective breeding were used in this study. All animals were bred and housed in the Animal Resource Center, to maintain constant environmental conditions. The animals were housed in large cages with no more than five rats per cage. Purina Lab Chow and water were provided ad libitum, and a 12-hr light/dark cycle was used, with the lights coming on at 6:00 A.M. The experiments reported here were carried out in accordance with the Declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals, as adopted and promulgated by the National Institutes of Health.

The experimental procedures and protocols used for determining the electrophysiological effects of EtOH on Purkinje neuron activity in situ have been previously described in detail (Palmer et al., 1987, 1992). Rats were anesthetized with urethane (1.25 g/kg i.p.), intubated and placed in a stereotaxic frame. Body temperature was monitored with a rectal probe and maintained at 37°C with a heating pad. After the skull and dura over the cerebellar vermis were removed, the cisterna was opened at the foramen magnum and the exposed surface of the cerebellum was covered with 2.4% agar in saline to reduce brain pulsation.

Spontaneous action potentials of single Purkinje neurons were recorded extracellularly from lobules VI and VII of the cerebellar vermis of each animal, using a 5 M NaCl-filled barrel of a two-barrel glass micropipette (Palmer et al., 1980). Purkinje neurons were identified both by their anatomical location and by their characteristic discharge pattern of single and complex spikes (Eccles et al., 1967). Single action potentials were monitored on an oscilloscope, separated from background activity and converted to constant-voltage pulses with a window discriminator. The spontaneous discharge rates were integrated over 1-sec epochs, displayed as action potentials per second (hertz) on a strip chart recorder (ratemeter record) and monitored on an Apple IIe computer as peri-event time histograms for subsequent data analysis.

EtOH (750 mM in 0.9% saline) was administered locally by pressure ejection from the drug barrel of the micropipette, as has been previously described in detail (Palmer, 1982). Previous studies have shown that drug administration with this technique is reproducible and linearly related to the pressure and the duration of the injection (Gerhardt and Palmer, 1987; Palmer et al., 1980, 1986; Stone, 1985). Thus, for ejection applications shorter than 90 sec, the dose of EtOH can be expressed as the product of the pressure (in pounds per square inch) and the duration of the ejection (in seconds). The response of each Purkinje neuron to EtOH was determined as the pressure ejection dose (pounds per square inch-second) of EtOH required to elicit an approximately 50% depression of the spontaneous firing rate. A 30 to 70% response window was used to avoid ceiling and threshold effects (Palmer, 1982; Sorensen et al., 1980). Each neuron was required to exhibit a stable firing rate during pre-EtOH and post-recovery control periods, and EtOH responses were acceptable only if they were either repeatable in the absence of tachyphylaxis or stable after the development of RANT. Previously described data acquisition strategies were used to minimize variability in neuronal EtOH exposure between micropipettes (Gerhardt and Palmer, 1987; Palmer et al., 1986). Each micropipette was used to apply EtOH to at least two Purkinje neurons in each rat, and at least two micropipettes were used to sample data from any one rat during a given recording session.

The data from the ratemeter records were digitized with a graphics tablet. These records and the peri-event time histograms collected on the Apple IIe computer were analyzed by computer for percentage response to EtOH, as previously described (Palmer and Hoffer, 1980). Previous reports have validated this approach for quantitative microadministration of drugs (Freedman et al., 1975; Palmer, 1982) and have shown that neuronal responses to locally applied drugs can be evaluated independently of variations in background discharge (Freedman et al., 1975). Controls for pressure ejection artifacts and solution osmolarity were used as previously described (Palmer, 1982; Palmer et al., 1986).

RANT to the depressant effects of locally applied EtOH on single Purkinje neurons was produced for these experiments using two distinct paradigms, 1) repeated lower dose applications of EtOH that initially elicit an approximately 50% depression of the neuronal firing rate of a given Purkinje neuron (fig. 1A) and 2) local application of a single high conditioning pressure dose of EtOH (fig. 1B). Before either RANT paradigm, initial neuronal sensitivity was investigated by determining a control pressure dose of EtOH that causes an approximately 50% depression of Purkinje cell firing rate, as described above. The initially effective pressure dose of EtOH was readministered as a "test dose" on some cells 90 to 120 sec after tolerance was established, to assess any attenuation of the EtOH-induced inhibition of neuronal firing rate (tachyphylaxis). After RANT was induced by one of the aforementioned methods, the dose of EtOH was increased and reapplied at 90- to 120-sec intervals until once again the firing rate of the Purkinje cell was depressed by approximately 50%. The change in dose to give that 50% response was used as an index of RANT. A neuron was categorized as having expressed RANT to EtOH if this dose was at least double the control dose that originally elicited a 50% depressant base-line response in the EtOH-naive Purkinje cell and/or if the cell showed tachyphylaxis in response to repeated EtOH applications.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Flow diagram illustrating the time courses of local EtOH applications that were used to study the production of RANT. A, repeated applications of test doses (small arrows) of EtOH, which initially caused 50% inhibition of neuronal activity, produced maximal acute tolerance (MT) after five to seven EtOH applications on LAS Purkinje neurons. B, one high conditioning dose (large arrow) of EtOH produced maximal acute tolerance on LAS Purkinje neurons already by the next test dose application (various time points along the dashed line). C, two test doses separated by 5 min did not cause maximal acute tolerance. D, three test doses over 4 min did not cause maximal acute tolerance. E, three test doses over 7 min did cause maximal acute tolerance in LAS rats. Note that acute tolerance was not observed for all neurons and was generally not observed in HAS rats. Initial EtOH sensitivity was determined by the first test dose on each time line.

The production of RANT by the repeated application of EtOH doses that caused 50% depressions of the cell under study was similar to that which we used previously to investigate this phenomenon (Palmer et al., 1992). Maximal RANT was produced by five to eight such EtOH applications. This paradigm did not involve EtOH doses in LAS rats higher than that required to cause 50% inhibitions, and HAS neurons were never exposed to the higher, tolerance-inducing EtOH doses studied in the LAS rats. For most experiments, the initially effective dose was repeated every 2 min (fig. 1A) until the neuron no longer exhibited a depression of its firing rate in response to EtOH application or until three subsequent EtOH pressure doses produced no further tachyphylaxis. In some experiments, the doses were separated by 5 min (fig. 1, C and E) or other time periods, as is indicated.

A single, high, conditioning pressure dose of EtOH (fig. 1B), which produced maximal neuronal tolerance in LAS rats, was studied to assess its effectiveness to produce RANT in HAS rats. Initially, conditioning doses between 150 and 300 psi-sec were investigated in LAS rats, because this range of doses was previously found to cause RANT in LAS Purkinje neurons (Palmer et al., 1987, 1992). We found that 270 psi-sec EtOH caused maximal RANT in the neurons that showed this response, and we used that "conditioning dose" of EtOH throughout the study.

Recovery from RANT to the depressant effects of EtOH was initially studied 5 and 20 min after its production. The time course of the development of and recovery from RANT was further characterized by repeating the initially effective EtOH test dose 2, 5, 10, 20 or 40 min after RANT induction. RANT for these experiments was calculated as the change in neuronal responsiveness to a local EtOH application from that caused by the initial application to that cell. Recovery from RANT in the initial two-time point study is expressed as a percentage of that initial EtOH response, whereas the results of the more detailed time course study are expressed as a percentage of the maximal RANT achieved in that neuron. The development of RANT in the latter experiment is expressed as a percentage of the acute tolerance produced by five EtOH test doses spaced at 2-min intervals.

Statistical significance was determined as indicated in "Results," using either two-tailed Student's t tests or one-way analysis of variance, followed by a Tukey-Kramer post hoc analysis if the analysis of variance was significant. The minimum n for a given experiment in this study was determined a priori, using a power calculation (Lachin, 1981), from previously published data (Palmer et al., 1992).

	Results

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Local applications of EtOH from multibarrel micropipettes depressed the firing of single cerebellar Purkinje neurons in both LAS and HAS rats, although higher EtOH doses were required to cause this effect in the LAS rats (fig. 2). Thus, the initial EtOH sensitivity of these neurons was 2.2-fold higher in HAS rats than in LAS rats before the development of any RANT, when compared between 18 Purkinje neurons in 5 LAS rats and 34 Purkinje neurons in 11 HAS rats (fig. 3A), and this difference was statistically significant (P < .0001; two-tailed Student's t test). These local pressure ejection applications of EtOH caused 46 ± 2% and 45 ± 3% depressions of neuron activity in HAS and LAS rats, respectively, which met our criterion of approximately 50% responses. Thus, the effects caused by the different effective EtOH doses between these two rat lines were not significantly different from each other (P > .1). RANT, expressed as tachyphylaxis of the responses to an originally effective test dose of EtOH, often developed after a single 270-psi-sec conditioning dose of EtOH (fig. 1B) in LAS rats but not HAS rats (fig. 2). This effect was expressed as a decreased maximal neuronal response to an EtOH dose, whereas the rate of recovery of neuronal firing with the EtOH dose was unchanged. When the test dose of EtOH was subsequently raised on these acutely tolerant cells to cause neuronal responses similar to those reported above, the EtOH sensitivity difference between the same HAS and LAS Purkinje neurons studied above increased to 3-fold (fig. 3B), even though only 50% of neurons sampled expressed desensitization to the EtOH effect. The primary change accompanying acute tolerance was a decrease in LAS neuron sensitivity to the depressant effects of EtOH, which is characterized in more detail below, whereas the EtOH doses required to depress HAS neurons were for the most part unaltered by the conditioning EtOH dose.

View larger version (44K): [in this window] [in a new window]   Fig. 2.   Sequential ratemeter records showing the development of RANT from a LAS neuron but not a HAS neuron. Firing rate, in action potentials per second (hertz), is represented on the vertical axis, and time is represented on the horizontal axis (see calibration bars at the end of each ratemeter record). EtOH was locally applied from a micropipette for the duration of each bar overlying the ratemeter records; the dose is indicated above each bar. A, a LAS neuron that originally required a 150-psi-sec pressure application of EtOH to cause an initial 36.5% depression (within the 30-70% target window) of neuronal firing rate. After a large conditioning dose of EtOH, RANT was observed to a second 150-psi-sec EtOH application, which caused only 17% depression. B, a HAS neuron that originally exhibited 42.5% depression of firing rate in response to a 40-psi-sec dosage of EtOH. After the neuron was exposed to a 270-psi-sec conditioning dose, a subsequent 40-psi-sec application depressed the cell's firing rate by 44.0%, exhibiting no development of RANT.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Bar graphs showing the sensitivity difference between LAS and HAS Purkinje neurons to locally applied EtOH before and after a 270-psi-sec conditioning dose of EtOH. A, LAS neurons (n = 18) were 2.2-fold less sensitive to EtOH (required a higher dose to cause depressions) than were HAS neurons (n = 34) before the development of acute tolerance. B, after the conditioning dose, which caused the development of acute tolerance in 50% of the LAS neurons sampled and represented here, the EtOH sensitivity difference between the same LAS and HAS Purkinje neurons increased to 3-fold. ***, significantly different, P < .0001; Student's t test.

RANT developed to five to eight repeated lower test doses of EtOH (fig. 1A), which initially caused roughly 50% depressions of neuronal firing, in 15 of the 30 Purkinje neurons studied in LAS rats. Similarly, a 270-psi-sec conditioning dose of EtOH (fig. 1B) induced RANT in 16 of the 38 LAS Purkinje neurons studied with this method (fig. 4). Local applications of EtOH caused an average of 41 ± 5% inhibition of neuronal activity before the development of RANT in these neurons, and this effect was significantly reduced (P < .0001) to an average of 8 ± 4% inhibition after the 270-psi-sec application of EtOH (fig. 5A). In addition, the average EtOH pressure application required to elicit an approximately 50% depression in the cell firing in these seven neurons was significantly increased (P < .0001) with the development of RANT to a single 270-psi-sec EtOH dose (fig. 5B). Consistent with our previous report (Palmer et al., 1992), the development of RANT to repeated test doses of EtOH (fig. 1A) was also associated with a similar increase in the EtOH dose required to cause 50% inhibitions of neuronal activity in the six neurons studied in such detail.

View larger version (39K): [in this window] [in a new window]   Fig. 4.   Bar graph illustrating the percentage of Purkinje neurons that developed RANT to EtOH in LAS and HAS rats by way of both the repeated-test dose and single-conditioning dose methods. The vertical axis represents the percentage of cells sampled, showing RANT after five to eight repetitions of EtOH test doses for 30 LAS and 28 HAS neurons, and after a single 270-psi-sec conditioning dose of EtOH for 38 LAS and 34 HAS neurons.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Bar graphs characterizing RANT to EtOH on seven LAS Purkinje neurons that showed RANT. A, before the development of RANT, local applications of EtOH caused an average of 41 ± 5% depression of the neuronal firing rate. After the development of RANT with application of a large conditioning dose (270 psi-sec), the same local applications of EtOH produced only an average of 8 ± 4% inhibition. This loss of the depressant effect of EtOH was statistically significant (***P < .0001; Student's t test). B, the development of RANT also caused the average dose of EtOH required to elicit a 50% depression in firing rate to increase significantly (***P < .0001, Student's t test) in these seven LAS neurons.

In contrast to our findings in LAS rats, RANT to the depressant effects of repeated test doses of EtOH (fig. 1A) developed in only 1 of 28 HAS Purkinje neurons studied, even when the test doses were repeated every 90 sec for up to 15 min. Similarly, the 270-psi-sec conditioning dose of EtOH (fig. 1B) caused RANT to these EtOH effects in only 2 of the 34 HAS Purkinje neurons investigated (fig. 4). The two neurons that showed RANT in this latter experiment exhibited a 57% depression of neuronal activity in response to initial EtOH applications, and this response was reduced to an average of 18% depression after the 270-psi-sec EtOH conditioning dose application.

When expressed in the above experiments, acute tolerance to locally applied EtOH was first observed within 90 sec after the 270-psi-sec EtOH application, was maximally developed within 7.5 min of that conditioning dose (fig. 1B) and was not further increased by subsequent repeated applications of the lower test dose of EtOH. Maximal RANT was also produced by five to eight of the lower test doses repeated every 2 min (fig. 1A), and this paradigm generally produced as much acute tolerance as the one 270-psi-sec conditioning dose described above. However, maximal RANT to the neuronal effects of EtOH appeared to take several minutes to develop. Thus, the time interval between test dose applications altered the time course of tolerance development. For example, the third of three test doses of EtOH, separated by 2 min (fig. 1D), showed only 37% of the acute tolerance that developed during five such EtOH applications in six LAS cells showing this response. However, 98% of the acute tolerance produced by five EtOH applications was already developed if the third response was separated from the second by 5 min (fig. 1E) instead of 2 min (fig. 6). In addition, the size of the conditioning dose influenced the degree of submaximal RANT developed. Thus, the depressant response of the second of two similar test doses of EtOH separated by 5 min (fig. 1C) was reduced by an average of only 28% in 15 LAS neurons. In contrast, the average EtOH response to a test dose applied 5 min after a 270-psi-sec conditioning dose of EtOH (fig. 1B) was reduced 5-fold in nine LAS neurons, compared with that observed before the conditioning dose (fig. 7). This represented a >90% development of maximal RANT in these cells.

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Bar graph depicting the time dependence of the maximal development of RANT to repeated EtOH applications. The ordinate represents the percentage of the acute tolerance caused by five similar local EtOH applications. Only 37% of the maximal RANT, which developed during five such local applications, was seen as a result of the third EtOH application when test doses of EtOH were separated by 2 min. When the spacing between the second and third doses was increased to a period of 5 min, 98% of maximal RANT was developed after the third local application of EtOH. **P < .01 (n = 6; Student's t test). Data are quantitated as the percentage of maximum RANT for each neuron.

View larger version (36K): [in this window] [in a new window]   Fig. 7.   Bar graph illustrating the dose dependence and recovery from RANT produced 5 min after two EtOH applications. The vertical axis represents EtOH responsiveness as a percentage of the response to an initial EtOH application that reduced spontaneous activity by 50%. A control dose of EtOH that caused an initial 50% depression of neuronal activity was followed 2 min later either by a second dose of the same size () (n = 15) or by a higher, 270-psi-sec conditioning dose () (n = 14). When applied 5 min after the second EtOH application, the response to a test dose of EtOH of the same size as the initial control dose showed less RANT (more effectively replicated the response to the initial control EtOH application) after a second control dose than was observed if the second EtOH application was the higher conditioning dose. These neurons completely recovered from the acute tolerance caused by either EtOH dose 20 min after the last EtOH application. Statistical comparisons were performed by analysis of variance followed by a Tukey-Kramer analysis. *P < .05, ***P < .001.

EtOH-induced depressions recovered to control values within 20 min of the production of RANT either by application of two lower test doses of EtOH alone to 15 LAS neurons or by a single 270-psi-sec conditioning application of EtOH to 14 LAS cells (fig. 7). Furthermore, 20 min was sufficient time for EtOH-induced depressions to show >90% recovery from maximal acute tolerance produced by repeated test doses of EtOH in an additional 16 LAS neurons (fig. 8). No recovery was observed from maximal acute tolerance 5 min after it was produced by repeated test doses of EtOH, and only partial recovery from RANT was observed 10 min after the last EtOH application (fig. 8).

View larger version (41K): [in this window] [in a new window]   Fig. 8.   Bar graph summarizing the time course for the recovery from RANT for 16 LAS Purkinje neurons that showed this phenomenon. The ordinate represents the percentage of maximal acute tolerance observed on a given neuron. No recovery was apparent at 5 min after maximal tolerance development via repeated test doses of EtOH. Only partial recovery occurred over a 10-min time period, and this was significantly different from the magnitude of recovery observed at the 20-min time point (P < .01). Furthermore, 20 min was sufficient for >90% recovery from maximal tolerance, which was also significantly different from that seen at the 5-min time point (***P < .001). Statistical comparisons were performed by analysis of variance followed by a Tukey-Kramer analysis.

	Discussion

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We previously found that RANT develops to repeated local EtOH applications that initially cause 50% depression of Purkinje neuron firing in SS mice (Sorensen et al., 1980) and LAS rats (Palmer et al., 1992). In the present investigation, a single, high, conditioning dose of EtOH caused a similar effect. This desensitization was characterized by both a decrease in neuronal responsiveness to a given EtOH dose and an increase in the EtOH dose required to cause 50% depressions of neuronal activity and was similar to that observed after repeated lower depressant doses of EtOH, both here and in our previous investigation (Palmer et al., 1992). A conditioning dose of EtOH that is high enough to cause maximal RANT in LAS Purkinje neurons produced no more RANT in HAS rats than did the repeated lower EtOH doses that evoke 50% depressions of neuronal activity in those animals. Thus, the low expression of RANT in HAS neurons is not the result of the lower EtOH doses used in these animals being too small to produce this phenomenon. Rather, we conclude that approximately 95% of HAS Purkinje neurons do not express RANT to the depressant effects of EtOH. In contrast, RANT makes a significant contribution to the acute insensitivity of LAS Purkinje neurons to the depressant effects of EtOH.

We found a significant difference between the initial EtOH sensitivity of LAS and HAS Purkinje neurons before the development of any RANT that was not present in generation 3 of these animals (Palmer et al., 1987). This differential sensitivity of LAS and HAS rats from generation 21 in the present study, however, was no larger than that found in generation 8 (Palmer et al., 1992). One difference between the present data and the study done with animals from generation 8 was that the previous study was performed on individual animals behaviorally selected for extreme EtOH sensitivity or insensitivity within their respective HAS and LAS populations, whereas the animals in the current study were randomly selected. Thus, in the previous study of generation 8 animals we may have sampled animals that express EtOH sensitivities that are more divergent than would be represented by average EtOH sensitivities in the current data. These data also suggest that the continued separation of LAS and HAS behavioral sensitivity to EtOH with selective breeding since generation 8 is mediated by phenotypes in addition to initial Purkinje neuron sensitivity.

The differential sensitivity of LAS and HAS Purkinje neurons to the acute actions of EtOH, however, is not mediated solely by the initial EtOH sensitivity of these neurons but is enhanced by the development of RANT. Acute tolerance increased the mean dose required to cause 50% responses in the population of LAS neurons sampled, even though not every LAS neuron sampled expressed RANT during EtOH exposure. Furthermore, the effective EtOH dose for producing depressions on neurons expressing RANT was higher for these LAS neurons from generation 21 than the value we previously reported for generation 8 (Palmer et al., 1992). Thus, an increase in the degree of RANT expressed by LAS neurons could contribute to the additional behavioral EtOH insensitivity that these animals have developed with continued selective breeding since generation 8.

Acute tolerance to the neuronal effects of EtOH develops within only a few minutes on neurons that show this phenomenon. Similarly to our previous report (Palmer et al., 1992), EtOH-induced depression desensitized with four or five repeated applications on most cells studied, and this occurred within 5 to 10 min of the initial EtOH dose. A similar degree of maximal tolerance developed to a single high conditioning EtOH dose within 7.5 min. This rapid onset of neuronal tolerance to EtOH in the cerebellum is consistent with older behavioral studies, which indicated that tolerance could develop during an initial EtOH dose (Goldstein, 1983; Laverty, 1989; Littleton, 1980; Mellanby, 1919). In addition, previous data suggest that rapidly acquired tolerance at least partially mediates the sensitivity difference to EtOH-induced ataxia in LS and SS mice (Keir and Deitrich, 1990; Parsons et al., 1982), as well as in alcohol-tolerant and alcohol-nontolerant rats (Le and Kiianmaa, 1989). Furthermore, Gill et al. (1993) have rotorod evidence suggesting that behavioral tolerance develops in SS mice within 3 to 5 min of EtOH exposure, and cellular tolerance to EtOH effects on GABA_A-mediated Cl flux has been previously reported to develop within 5 min of the initial EtOH exposure (Allan and Harris, 1987). Thus, as originally pointed out by Goldstein (1989), acute tolerance that develops over a period of a few minutes may be difficult to distinguish from low initial sensitivity in studies of systemic EtOH administration.

The development and maintenance of RANT is dose-dependent as well as time-dependent. A single EtOH test dose causing 50% depressions does not cause as much neuronal tolerance as does the higher conditioning dose used in this study, and repeated applications of the lower test doses are required to produce as much tolerance as does one higher conditioning dose. However, repeated test doses do not produce any more tolerance than does the higher conditioning dose, suggesting that this phenomenon is not simply the product of repeated EtOH depressions. RANT takes several minutes to maximally develop, even though some tolerance was first observed only 90 sec after an initial EtOH application. Thus, maximal desensitization of the EtOH response is not caused either by two EtOH test doses separated by 5 min (5 min between the first and last doses) (fig. 1C) or by three such EtOH test doses separated by 2 min (4 min between the first and third EtOH exposures) (fig. 1D). However, RANT is maximally developed by three EtOH applications when a total of 7 min is allowed between the first and last EtOH exposures (fig. 1E). These data suggest that 5 to 7 min are required for development of maximal RANT, regardless of the number of test doses of EtOH applied during that period. Our observation that RANT requires a few minutes to develop, together with the finding that neurons expressing acute tolerance require 20 min to recover from this EtOH desensitization, suggests the involvement of processes that are slower than direct interactions with membrane function, which should require only milliseconds. Second-messenger mechanisms, which are involved in post-translational regulation of various membrane components, are more likely the target of this EtOH action and have been implicated in the beta adrenergic mechanism of acute EtOH actions in the cerebellum and other brain areas (Bode and Molinoff, 1988; Freund and Palmer, 1996; Hoffman and Tabakoff, 1990; Luthin and Tabakoff, 1984).

In conclusion, the EtOH insensitivity of LAS Purkinje neurons is mediated not only by low initial sensitivity to the depressant effects of EtOH but also by the development of RANT to this neuronal effect over the first few minutes of EtOH exposure. The selective expression of this phenomenon contributes to the differential sensitivity of LAS and HAS rats to the depressant effects of EtOH on these neurons; the characterization, in the present report, of conditions under which RANT to EtOH is expressed allows subsequent investigations of the mechanisms mediating this desensitization. Indeed, we recently collected preliminary data suggesting that RANT to cerebellar EtOH effects is prevented by timolol (Pearson et al., 1996), a beta adrenergic antagonist, and that the beta adrenergic mechanism of EtOH action, which we previously described in cerebellum (Lee et al., 1995; Lin et al., 1993, 1994), is more active in LAS rats than in HAS rats (Donatelli, et al., 1995). At present, however, the mechanisms mediating RANT and the role of this acute tolerance mechanism in other EtOH actions are not understood.